A key compensatory response in the stressed heart is myocyte hypertrophy. Although cardiac hypertrophy can maintain cardiac output in response to elevated wall stress, sustained cardiac hypertrophy is often accompanied by maladaptive remodeling which can ultimately lead to heart failure. Recent evidence suggests that p90 ribosomal S6 kinase (RSK) might be an important regulator of cardiac myocyte hypertrophy. My proposed studies will further our understanding of the role of RSK3 in cardiac remodeling and help determine whether RSK3 would make an attractive therapeutic target for the attenuation of maladaptive hypertrophic signaling. Specific Aim 1: Characterization of a RSK3 binding site within a cardiac myocyte multimolecular signaling complex. I propose to refine the mapping of the predominant RSK3 binding site in mAKAPp and to test whether RSK3 binds to mAKAPp directly using expression in mammalian heterologous cells and bacteria. Specific Aim 2: Studies determining RSK3-dependent NFATc function in myocytes. A potential mechanism by which RSK3 may transduce hypertrophic signaling is through the phosphorylation of the pro-hypertrophic NFATc (nuclear factor of activated T-cells) transcription factor family. I will test whether RSK3 can phosphorylate and activate NFATc family members in neonatal rat cardiac myocytes under conditions that elicit cardiac myocyte hypertrophy in vitro. The effect of RSK3-dependent phosphorylation on NFATc function will be measured using luciferase reporter assays. In addition, I will use mAKAPp RSK3-binding site mutants to test whether RSK3 association with the mAKAPp signalosome is required for NFATc activity and myocyte hypertrophy. Specific Aim 3: The requirement for RSK3 in cardiac hypertrophy in vivo. In order to test whether RSK3 is required for myocyte hypertrophy, I will characterize the phenotype of cardiac-specific RSK3 knock-out mice that have been previously generated. I will challenge the RSK3 mice by left coronary artery ligation and induction of myocardial infarction. Cardiac hypertrophy will be analyzed at one month post-infarction by echocardiography and hemodynamic analysis and by gross and histopathology. Further, in order to address my hypothesis that RSK3 phosphorylation of NFATc isoforms is important for hypertrophy, NFATc activity will be monitored in vivo by mating the cardiac-specific RSK?''mice to NFAT-luciferase indicator mice. Cardiac hypertrophy is a leading risk factor for heart failure, and heart failure is a syndrome of major public heath significance affecting 5.2 million US individuals. A better understanding of the mechanisms controlling myocyte hypertrophy may allow for better therapeutic regimens with decreased mortality.